The biological function of a protein is dependent on its three dimensional structure. Proteins are formed as linear chains of amino acids, termed polypeptides. In vivo, in the appropriate conditions within the cell, the linear polypeptide chain is folded, a process which may be assisted by proteins called chaperones. A mature folded protein has an active three dimensional conformation known as the native structure. The structure depends on weak forces such as hydrogen bonding, electrostatic and hydrophobic interactions. These forces are affected by the protein's environment, so changes in the environment may cause structural disruption resulting in denaturation and/or degradation of the protein and loss of function. Such problems are encountered during processing of proteins and on storage of proteins.
The production of proteins by genetic engineering often results in the accumulation of non-active protein aggregates as inclusion bodies. After isolation and purification from the host cells, proteins or inclusion bodies have to be unfolded (denatured) and subsequently refolded (renatured) so that the proteins regain their native structure and bioactivity.
Traditional protein folding methods involve denaturant dilution or column-based approaches. Denatured protein is commonly refolded by diluting the denaturant. This induces a hydrophobic collapse of the protein molecule and in doing so the protein shields its hydrophobic patches in the core of the molecule. Unfortunately, on hydrophobic collapse, proteins do not always form the native bioactive conformation, two competing reactions occur: refolding and aggregation. It is suggested that a driver for protein aggregation is hydrophobic amino acid residues exposed at the surface of the molecule. Aggregation is undesirable and reduces the yield of functional, native protein. Refolding is known as a first order reaction (Rate=K*[prot]), but the aggregation reaction is favoured over refolding at high concentration as it is a higher order reaction (n) (Rate=K′*[prot]n). The proportion of protein refolding:protein aggregation is strongly dependent on the protein concentration. At process scale this concentration dependency can result in increased aggregation, and thus reduced refolding yield. This can be due to imperfect mixing patterns in the protein solution. Processing large protein molecules is particularly difficult as they diffuse more slowly than the smaller denaturant molecules, thus creating micro-environments with high localised protein concentrations and low denaturant concentration, that is, an environment that favours protein aggregation over protein refolding.
For proteins with disulphide bonds the native protein often needs to be “matured”; during maturation, non-aggregated monomeric protein molecules with non-native disulphide bonds may be created initially; then, using a protein specific redox couple, the disulphide bonds shuffle and the protein matures to a functional protein molecule with native disulphide bonds.
Refolding processes usually involve dispersing the denatured protein molecules in a buffer in the presence of “refolding aids” to enhance renaturation. Folding aids usually increase the solubility of the folding intermediates and/or change the relative reaction rates of the folding and aggregation reactions. Polyethylene glycol and various sugars, e.g. sucrose, glucose, N-acetylglucosamine, and detergents, e.g. Chaps, Tween, SDS, Dodecylmaltoside have been employed as refolding aids (De Bernadez Clark (1998) Current Opinion Biotechnol. 9, 157-163).
α, β and γ cyclodextrins (CDs) have been reported to be useful in stabilisation, solubilisation and affinity purification of certain enzymes, but both the nature of the interactions between these CDs and proteins, and their effect on bioactivity remain unclear. α, β and γ-cyclodextrins have been used as artificial chaperones to aid protein refolding in both detergent-free (Sharma et al, EP 0 871 651, U.S. Pat. No. 5,728,804) and detergent-containing refolding environments (Gellman & Rozema, U.S. Pat. No. 5,563,057). Cyclodextrins are cyclic oligosaccharides composed of multiple glucose residues. They are classified according to the number of sugar residues within the ring structure, α-cyclodextrin has 6 glucose residues, α-cyclodextrin has 7 glucose residues and γ-cyclodextrin has 8 glucose residues. Cyclodextrins can be modified by derivatisation to produce derivatives.
The inner cavity of cyclodextrins is hydrophobic whereas the outer surface is hydrophilic. The hydrophobic interior is capable of encapsulating poorly soluble drugs. The hydrophilic exterior assists in solubilisation, so cyclodextrins are useful adjuncts in pharmaceutical formulation.
EP 0 094 157 & U.S. Pat. No. 4,659,696 (Hirai et al) describe the use of α-, β-, and γ-cyclodextrin derivatives in pharmaceutical compositions consisting essentially of a physical mixture of a hydrophilic, physiologically active (folded, native) peptide and a cyclodextrin derivative, the composition being a uniform mixture in dosage form.
EP 0 437 678 B1, U.S. Pat. Nos. 5,730,969 and 5,997,856 (Hora) describe methods for the solubilisation and/or stabilisation of polypeptides, especially proteins, using specified cyclodextrin derivatives: hydroxypropyl, hydroxyethyl, glucosyl, maltosyl, and maltotriosyl derivatives of β- and γ-cyclodextrin; the hydroxypropyl-β-cyclodextrin derivative being preferred. Also disclosed are aqueous and lyophilised compositions comprising a polypeptide, optionally a protein, and the above specified cyclodextrin derivatives.
EP 0 871 651 & U.S. Pat. No. 5,728,804 (Sharma et al) are concerned with a method for renaturing an unfolded or aggregated protein in a detergent-free aqueous medium with an amount of a cyclodextrin effective to renature said unfolded or aggregated protein. In this instance, the protein is present at a low concentration selected to minimise aggregation, preferably at around 0.05 mg/ml, and spontaneously refolds in a refolding buffer containing cyclodextrin. After refolding the cyclodextrin is removed by dialysis.
U.S. Pat. No. 5,563,057 (Gellman & Rozema) describes a method for refolding an enzyme from a misfolded configuration to a second native active configuration by adding a detergent having a linear alkyl non-polar portion, e.g. CTAB and Triton®-X 100 (Octoxynol-9), to misfolded enzyme to form an enzyme-detergent complex, which is then contacted with a cyclodextrin to allow the enzyme to assume the second active conformation.
Hinrichs et al (2001). International Journal of Pharmaceutics, 215, 163-174, describes the use of non-derivatised inulins, inulin SC 95 (DPn/DPw=5.5/6.0), inulin RS (DPn/DPw=14.2/19.4), and inulin EXL 608 (DPn/DPw 23.0/26.2) to protect alkaline phosphatase from degradation during freeze drying and subsequent storage of the dried protein.
WO 96/41870 (Gombac et al) describe frozen, dried or lyophilised hydrosoluble collagenase compositions containing isomalt and/or inulin (non-derivatised) to stabilise the collagenase.
To date, inulins have not been reported to be useful as protein folding aids. Inulins are D-fructans, generally consisting of chains of polyfructose in which the fructose units are connected to each other mostly or exclusively by .beta.(2-1) linkages. Inulin occurs in nature, in general, as a polydisperse mixture of polyfructose chains, most of which have a glucosyl unit at one terminus. Inulin can be obtained from bacterial syntheses, extracted from plants or can be made in vitro by enzymatic synthesis starting from sucrose. Inulin produced by bacteria is more branched than inulin from plant origin and commonly has a higher molecular weight (ranging from about 2,000 up to about 20,000,000), whereas inulin from plant origin is generally composed of linear or slightly branched polyfructose chains or mixtures thereof with a molecular weight commonly ranging from about 600 to about 20,000.
Inulin can be represented, depending from the terminal carbohydrate unit, by the general formulae GF.n or F.n, wherein G represents a glucosyl unit, F a fructosyl unit, and n is an integer representing the number of fructosyl units linked to each other in the carbohydrate chain. The number of saccharide units (fructose and glucose units) in one inulin molecule is referred to as the degree of polymerisation, represented by (DP). Often, the parameter (number) average degree of polymerisation, represented by (DP), is used too, which is the value corresponding to the total number of saccharide units (G and F units) in a given inulin composition divided by the total number of inulin molecules present in said inulin composition, without taking into account the possibly present monosaccharides glucose (G) and fructose (F), and the disaccharide sucrose (GF). The average degree of polymerisation (DP) can be determined, for example, by the method described by L. De Leenheer (Starch, 46 (5), 193-196, (1994), and Carbohydrates as Organic Raw Materials, Vol. III, 67-92, (1996)).
Inulin is commonly prepared from plant sources, mainly from roots of Chicory (Cichorium intybus) and from tubers of Jerusalem artichoke (Helianthus tuberosus), in which inulin can be present in concentrations of about 10 to 20% w/w of fresh plant material. Inulin from plant origin is usually a polydisperse mixture of linear and slightly branched polysaccharide chains with a degree of polymerisation (DP) ranging from 2 to about 100. In accordance with known techniques, inulin can be readily extracted from said plant parts, purified and optionally fractionated to remove impurities, mono- and disaccharides and undesired oligosaccharides, in order to provide various grades of inulin, e.g. as described in EP 0 769 026 and EP 0 670 850.
Inulin is commercially available, typically with a (DP) ranging from about 6 to about 40. Inulin from chicory is for example available as Inutec®N25 and RAFTILINE® from ORAFTI, (Tienen, Belgium) in various grades. Typical RAFTILINE® grades include RAFTILINE® ST (with a (DP) of about 10 and containing in total up to about 8% by weight glucose, fructose and sucrose), RAFTILINE® LS (with a (DP) of about 10 but containing in total less than 1% by weight glucose, fructose and sucrose), and RAFTILINE® .RTM HP (with a (DP) of at least 23, commonly with a (DP) of about 25, and virtually free of glucose, fructose and sucrose).
Inulins with a lower degree of polymerisation, usually defined as a (DP)<10, are commonly named inulo-oligosaccharides, fructo-oligosaccharides or oligofructose. Oligofructose can be obtained by partial (preferably enzymatic) hydrolysis of inulin and can also be obtained by enzymatic in vitro synthesis from sucrose according to techniques which are well-known in the art. Several grades of oligofructose are commercially available, for example as RAFTILOSE® from Orafti, (Tienen, Belgium), e.g. RAFTILOSE® P95 with a mean content of about 95% by weight of oligofructose with a degree of polymerisation (DP) ranging from 2 to 7 and containing about 5% by weight in total of glucose, fructose and sucrose. Inulins derivatised with hydrophobic alkyl chains on the polyfructose backbone are commercially available, for example Inutec® SP1 (SP1) from Orafti (Tienen, Belgium).
Various inulin derivatives and methods for the preparation of inulin derivatives are described in U.S. Pat. No. 6,534,647 (Stevens et al), the entire contents of which are incorporated herein by reference.
Starch is a well-known carbohydrate that is abundantly present in many plants as a biodegradable reserve polysaccharide. Starch molecules are polymers composed of D-glucosyl units which are linked to one another by α-1,4 glucosyl-glucosyl bonds, thus forming a linear chain starch structure (termed amylose) or by α-1,4 and α-1,6 glucosyl bonds thus forming a branched chain starch structure (termed amylopectin) having a α-1,6 glucosyl-glucosyl bond at the branching point. Starch occurs in nature as a polydisperse mixture of polymeric molecules which have, depending on the plant source, mainly a linear structure or mainly a branched structure. Starch can also occur in nature as a polydisperse mixture of molecules with said structures. The degree of polymerisation (DP), i.e. the number of glucosyl units linked to one another in a starch molecule, may widely vary and it depends largely on the plant source and the harvesting time.
The linkages between the glucosyl units are sensitive to hydrolysis, heat and shearing forces. This phenomenon is industrially exploited to prepare various starch derivatives, generically termed herein starch hydrolysates, through acidic hydrolysis, enzymatic hydrolysis, thermal treatment or shearing, or through combinations of said treatments. Depending on the source of the starch, the hydrolysis catalyst, the hydrolysis conditions, the thermal treatment and/or the shearing conditions, a wide variety of starch hydrolysates can be obtained, ranging from a product essentially composed of glucose, over products commonly termed glucose syrups, to products commonly termed maltodextrins and dextrins. Starch hydrolysates are well known in the art.
D-glucose (dextrose) presents strong reducing power. Starch hydrolysates are polydisperse mixtures, composed of D-glucose, oligomeric (DP<10) and/or polymeric (DP>10) molecules composed of D-glucosyl chains, which also present reducing power resulting from the presence of D-glucose and reducing sugar units (which are essentially terminal glucosyl units) on the oligomeric and polymeric molecules.
As a result, starting from a given starch product, the greater the extent of the hydrolysis, the more molecules (monomeric D-glucose, oligomeric and remaining polymeric molecules) will be present in the hydrolysate, and thus the higher the reducing powder of the starch hydrolysate obtained. Accordingly, the reducing power of starch hydrolysates has become the distinguishing feature of choice to differentiate and designate the various starch hydrolysate products. The reducing power is expressed as dextrose equivalents (D. E.) which formally corresponds to the grams of D-glucose (dextrose) per 100 grams of dry substance. D-glucose having by definition a D. E. of 100, the D. E. indicates the amount of D-glucose and reducing sugar units (expressed as dextrose) in a given product on a dry product basis. Thus the D. E. is in fact also a measurement of the extent of hydrolysis of the starch and also a relative indication of the average molecular weight of the glucose polymers in the starch hydrolysate.
The D. E. of starch hydrolysates, apart from hydrolysates composed essentially of D-glucose, may range from 1 to about 96 and starch hydrolysates are commercially available in a wide variety of grades based on the D. E.
Hydrolysates with a D. E. greater than 20 are commonly termed glucose syrups. Glucose syrups with a D. E. up to 47 can be dried by conventional techniques, for example by spray drying, to yield so-called “dried glucose syrups” in powder form, containing a maximum of about 5 wt % humidity.
Hydrolysates with a D. E. of 20 or less are commonly termed maltodextrins and dextrins. The manufacturing process usually involves a spray drying step at the end, yielding these hydrolysate products in powder form also containing a maximum of about 5 wt % humidity (wt % indicates % by weight).
Glucose syrups, maltodextrins and dextrins are made industrially at large scale from various starch sources under controlled hydrolysis conditions according to well-known methods. The various grades of starch hydrolysates obtained are usually defined by their starch source material and by their D. E. value, often in combination with an indication of the method of manufacture (e.g. maltodextrins/dextrins).
Although following certain Regulations the term “maltodextrins” is reserved to designate products derived from corn starch, the term maltodextrin(s) used herein is not limited to hydrolysates of corn starch, but indicates herein starch hydrolysates with a D. E. of 20 or less obtained from starch from any source.
Typical commercial sources of starch are corn, potato, tapioca, rice, sorgum and wheat. However, the starch hydrolysates suitable for use in connection with the present invention are not limited to starch from said sources, they extend to starch hydrolysates obtained from starch from any source.
Glucose syrups, maltodextrins and dextrins are well known and commercially available. For example, the production, properties and applications of glucose syrups and maltodextrins have been described in review articles in the book Starch Hydrolysis Products, Worldwide Technology, Production and Applications, Weinheim VCH Publishers Inc. (1992). Furthermore, in the technical brochure “GLUCIDEX Brochure 8/09.98” from the company Roquette, maltodextrins and dried glucose syrups are described and various grades are offered for sale.
There is a need for methods for the efficient preparation of correctly folded, non-aggregated, active protein, particularly for proteins produced using recombinant techniques. Control of folding and aggregation of proteins during processing and on storage is a recognised problem in many industries, in particular the pharmaceutical and biotechnology industries. The problems encountered with proteins may make manufacture of proteins difficult, result in low yields and render processes uneconomic, Methods, consumables, reagents and kits that permit control of protein refolding and modulate protein aggregation are commercially important. It is an object of the present invention to provide methods of protein folding, such that the disadvantages associated with present methods are alleviated.
Problems addressed by the present invention include reducing protein aggregation and achieving control of protein refolding, in particular of folding protein from a stable non-native state to a partially or fully folded state in a controlled manner to reduce aggregation and thereby increase the yield of soluble intermediates or native protein.